CN101912387B - Application of benzyl-containing flavonoid lignan in preparation of medicament for treating hepatitis B - Google Patents
Application of benzyl-containing flavonoid lignan in preparation of medicament for treating hepatitis B Download PDFInfo
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Abstract
The invention relates to application of benzyl-containing flavonoid lignan in preparation of a medicament for treating hepatitis B, in particular to application of flavonoid lignan and pharmaceutically-acceptable salt thereof in preparation of the medicament for eliminating hepatitis B e antigen, inhibiting hepatitis B virus desoxyribonucleic acid (HBV DNA) replication and treating viral hepatitis B. The flavonoid lignan can inhibit the activity of hepatitis B virus e antigen (HBeAg); the inhibition intensity of the flavonoid lignan under low concentration of 20 micrograms per milliliter is much higher than that of a positive control front-line medicament lamivudine and an interferon; and the compound with the concentration of 20 micrograms per milliliter displays an inhabitation ratio over 50 percent on the HBV DNA, so that the application of the flavonoid lignan in preparation of the medicament for eliminating the hepatitis B e antigen and the medicament for inhibiting the HBV DNA replication and treating hepatitis B viral diseases can be anticipated.
Description
Technical field
The present invention relates to medical technical field; Particularly; The present invention relates to the flavanolignan shown in the formula (1) or its officinal salt is used for preparation removing hepatitis B virus e antigen, suppresses the purposes that HBVDNA duplicated, treated the hepatitis B medicine; This flavanolignan finds to have strong effect through the pharmacodynamics test and suppresses the hepatitis B virus e antigen activity; And its inhibition strength under low concentration 20 mcg/ml just far above positive control one line medication lamivudine and-interferon; Simultaneously this chemical compound also demonstrates the suppression ratio greater than 50% to HBV DNA during 20 mcg/ml, thereby can expect and develop into the medicine that reduces the hepatitis B virus e antigen medicine, suppresses HBV dna replication dna or treatment hepatitis B virus infection disease.
Background technology
Hepatitis B (hepatitis B) is the infectious disease that is caused by hepatitis b virus hbv.HBV is a member of Hepadnaviridae hepadnaviridae, and it is shaped as the spheroidal particle of diameter 42 nanometers.HBV is peculiar virus, and less being infectious in other animal has only in human body or primate chimpanzee body and just can duplicate.This virus is propagated through hepatitis B virus carriers and hepatitis B patients'blood, saliva, seminal fluid, vaginal secretions, has chronic carrier state.Primary disease is widely current in China, and because of it has in vertical transmission, horizontal transmission, the family propagation, iatrogenic infection and multiple mode such as spread through sex intercourse, high to the crowd infection rate, infection rate reaches more than 35% in certain areas.According to interrelated data, hepatitis detects male patient and has reached 1.89 hundred million, and the number (carrier) nearly 400,000,000 of should not going to a doctor is one of the most serious infectious disease of current harm people ' s health.Hepatitis B clinical manifestation variation is prone to develop into chronic hepatitis B (CHB) and liver cirrhosis, and few patients can change primary hepatocarcinoma into.
Hepatitis B virus e antigen HBeAg is the structural protein of hepatitis B viruses (HBV) kernel, when hepatitis B virus is bred, produces in a large number.Hepatitis B viruses (HBV) has genome (only 3.2kb) minimum in all known dna viruses; Its gene five kinds of albumen (S, C, E, P, X) of mainly encoding; Wherein C albumen is viral core protein, and the E albumen proteic part that is C becomes hepatitis B virus e antigen (HBeAg); It is to have encoded but unassembled albumen in the virion can be secreted in the blood samples of patients when virus replication and go.Clinically, usually serum HBeAg is replied the important symbol thing of estimating as hbv replication, infectiousness, the degree that is in a bad way and to its treatment.This antigen and HBV DNA are closely related, are to express the very practical blood serum designated object of virus replication clinically.In its body of blood serum designated object HBeAg positive patient explanation hbv replication is arranged, so higher infectiousness is arranged, it is strong more that patient HBeAg expresses this patient's infectiousness of high more explanation.In like manner, suppress the secretion of HBeAg and duplicate also be in the research and development anti-hepatic-B virus medicine important target with detect target.HBeAg removes in the explanation body has lasting HBV to suppress, and ALT is normal, and the tissue inflammation necrosis alleviates, and the liver cirrhosis odds reduces.Therefore, serum HBeAg is believed to reflect more stable therapeutic effect, and HBeAg serum clear flag patient's immune system and begun to play a role.2002, deliver the researcher of result of study at " New England Journal of Medicine " and think: for CHB patient, remove as before liver cirrhosis, obtaining HBeAg, then its liver cirrhosis and hepatocarcinoma incidence rate will reduce by 10 times.All HBeAg serum is removed as one of treatment terminal point determining standard in the guide of the AASLD of U.S. hepatopathy research association, the Asia-Pacific liver APASL of research association and the EASL of EASL.So, can suppress, reduce the active drug that HBeAg medicine active or that express also promptly belongs to treatment hepatitis B virus infection disease.
At present, the medication to hepatitis B patient mainly is divided into several big type of the liver protecting and ALT lowering, antiviral, anti-hepatic fibrosis and adjusting immunity etc.The research that still is the antiviral treatment aspect that made progress in the last few years.Antiviral is a basic method, and the liver protecting and ALT lowering is an auxiliary treatment, takes stopgap measures and rarely seen effecting a permanent cure more.Yet; Can only reach for the antiviral clinically therapeutic scheme of viral hepatitis B at present and suppress hbv replication and secondary infection; Main medicine is still nucleoside medicine such as lamivudine (3-TC), Entecavir, adefovirdipivoxil (ADV), Sebivo etc., is in the interim emtricitabine of clinical trial, tenofovir, clevuding etc. in addition.In China, lamivudine has become the medical insurance medication, is applied to large quantities of HBV patients.Nucleoside medicine part advantage is: bioavailability is high, and is oral safer.Yet, though their disease controlling, fetch long prices first effectively; Second life-time service all drug resistance can occur, and the bounce-back in various degree that occurs HBV DNA, ALT and liver histological after the drug withdrawal; The 3rd, comparatively significantly ill effect, for example kidney injury, baby's teratogenesis etc. that the life-time service nucleoside medicine occurs.Headache is the most: virus is drug-fast cure rate to occur greatly reducing; Because nucleoside medicine is reversible to virus replication, thus to most of patient if desire to reach greatest treatment efficacy, the course of treatment must be more than 1 year; So its drug resistance occurs thereupon, does not just reach the effect of expection.In addition, nucleoside medicine be difficult in addition remove cccDNA, treatment after 1 year HBsAg be difficult to weak points such as cloudy commentaries on classics.
The biological engineering class antiviral drugs that interferon (α, beta-interferon) and recombinant interferon class etc. derive from the HL becomes research and treatment chronic viral hepatitis B CHB focus medicine in the recent period, and it has antiviral and immunomodulating dual function.Thereby it both can suppress virus replication through antivirus action and alleviate the liver cell inflammatory reaction, reduces hepatocellular damage, plays and improves patients clinical symptom and liver physiological function thereby delay PD; Again can the enhance immunity effect, through the effect of natural killer cell in the intensive aspect and helper T lymphocyte, especially can promote killer T cell to go to kill and wound by virus infected cell, therefore play antivirus action indirectly.The HBeAg serology conversion that interferon therapy CHB patient obtains is more more lasting than lamivudine, and " digestive tract " magazine the next item up research in 2003 shows: 3 years relapse rates of interferon therapy group HBeAg significantly are lower than the lamivudine group; And long-acting interferon can use weekly once, and is comparatively convenient.Therefore, interferon day by day becomes one of choice drug that is used to treat chronic viral hepatitis B virus clinically, but that its side effect and untoward reaction are reported is more, and total effective rate is not high, costs an arm and a leg, and patient economy burden is big, thereby causes and be difficult to clinically be widely used.
The basic link of another one that the inhibition hepatitis B virus is bred in vivo is to suppress duplicating of HBVDNA.The reduction of HBV dna level or be lower than detection level be the check antiviral drugs other one JINYAOSHI.So, Asia-Pacific liver EASD and European liver EASD all with the HBV DNA detection less than as one of hepatitis B virus patient treatment terminal point.Also tested compounds is regarded as one of the test event that to accomplish in China's new drug development guide for the inhibition strength of HBV DNA.Why lamivudine can become first-selected nucleoside medicine is because it has the activity of the inhibition HBV dna replication dna of strong effect.Therefore, can suppress the novelty medicine that chemical compound that the HBV dna replication dna can suppress HBeAg again will more promise to be the treatment hepatitis B.
Mandatory declaration be: the antiviral drugs of using at present is the inhibitor of virus replication just in fact, directly kill virus or break virus body, otherwise will damage host cell.These antiviral drugs (being mostly nucleoside medicine) also exist above-mentioned toxic and side effects big, be prone to cause and be prone to shortcoming such as knock-on after viral gene sudden change, the drug withdrawal.Therefore, the development of new antiviral drugs is the task of top priority in current medicament research and development field, and it all has extremely important social meaning and economic implications for treatment China a large amount of hepatitis B patient and virus carrier, the control source of infection etc.So new non-nucleoside hepatitis B virus inhibitor and this type of lead compound that can reduce HBeAg and/or inhibition HBV dna replication dna of discovery has very big guiding significance from the natural drug of national folk life-time service, and vast development prospect is arranged.
Based on this purpose; The inventor once accomplished the patent and the article of multinomial anti-hepatitis virus natural product and structure of modification derivant thereof in the past; Having found that multiple inhibition hepatitis B virus e antigen HBeAg is active or suppress the chemical compound of HBV dna replication dna, is feasible [referring to " medical usage of one type of mapping eucalyptus globulus alkanol type sesquiterpene for inhibiting hepatitis virus " (Zhao Yu, Liu Guangming, Yu Rongmin, Li Haibo etc. thereby explanation filters out the novelty medicine that suppresses hepatitis B virus e antigen, control hepatitis B virus infection from natural product and synthesis of derivatives thereof; ZL 200610053827.4); " medical usages of 2 beta-hydroxy Ilicis Purpureae acid for inhibiting hepatitis B virus " (Lee school Kun, Zhao Yu, Huang Kexin, Li Haibo etc.; ZL 200610053749.8); " 2 α, the medical usage of 3 beta-dihydroxies-5,11 (13)-diene eucalyptus globulus alkane-12-acid for inhibiting hepatitis B virus " (Zhao Yu, Zhang Li and, Dong Sun Han, Li Haibo etc.; ZL 200610053601.4); The purposes and the pharmaceutical composition thereof of hepatitis B virus " the eremophilane lactone suppress " (Zhao Yu, Li Haibo, Yang Leixiang, girth are new etc.; ZL03153691.3); " a kind of Eremophilone lactones acid natural product and application thereof " (Zhao Yu, girth are new, Shi Shuyun, Wang Xiaoyu etc.; ZL 200610053575.5); " a kind of eudesmane type sesquiterpenes acid and uses thereof " (Zhao Yu, Liu Guangming, Li Haibo, Wu Xiumei etc.; ZL200610053579.3); " purposes of laggera plant abstract in inhibiting herpes simplex virus and hepatitis B virus " (Zhao Yu, girth are new, Yu Rongmin, Bai Hua; CN 1989989A); " medical usage of 1 β-oxo-5,11 (13)-diene eucalyptus globulus alkane-12-acid for inhibiting hepatitis B virus " (Zhao Yu, Lee school Kun, Huang Kexin, Li Haibo etc.; CN 1927197A); " medical usage of 1 beta-hydroxy Ilicis Purpureae acid for inhibiting hepatitis B virus " (Zhao Yu, Lee school Kun, Huang Kexin, Wu Xiumei etc.; CN
1935131A); " mapping eremophilane acid and suppress the medical usage of hepatitis B surface antigen " (Huang Kexin, Lee school Kun ... Wang Xiaoyu, Zhao Yu etc.; CN 101239054); " 1-oxygen-substituted benzene formyl quinic acid chemical compound and suppress hepatitis B virus purposes " (Lee school Kun, Hu Lihong ... Wu Xiumei, Zhao Yu etc.; CN 101293836); Inhibition HBeAg that the inventor has delivered and HBV DNA isoreactivity article are referring to " In Vitro Antiviral Activity ofThree Enantiomeric Sesquiterpene Lactones from Senecio SpeciesAgainst Hepatitis B Virus ", Haibo Li (Li Haibo), Changxin Zhou (girth is new); ... Xiumei Wu (Wu Xiumei); Yu Zhao* (Zhao Yu), Antiviral Chemistry&Chemotherapy, 2005; 16,277-282; " Evaluation of Antiviral Activityof Compounds Isolated from Ranunculus sieboldii Miq.and Ranunculussceleratus L ", Haibo Li (Li Haibo), Changxin Zhou (girth is new); ... XiumeiWu (Wu Xiumei); Yu Zhao* (Zhao Yu), Planta Medica, 2005; 71 (12), 1128-1133; " Application of high-speed counter-current chromatographyfor the isolation of antiviral eremophilenolides from Lihulariaatroviolacea ", Shi, Shu-Yun (Shi Shuyun); Hai-Bo (Li Haibo) ... Zhao, Yu (Zhao Yu); Biomedical Chromatography; 2008,22 (9), 985-991; " Purification and identification of antiviral components from Laggerapterodonta by high-speed counter-current chromatography "; Shuyun Shi (Shi Shuyun); ... Yu Zhao (Zhao Yu) etc., Journal of Chromatography B, 2007; 859,119-124].Undoubtedly, continuing from natural product and structure of modification derivant thereof, to seek the lead compound that can remove HBeAg or inhibition HBV dna replication dna is that very therefore being necessary property is also classified as one of great special project of new drug development by the Ministry of Science and Technology with urgent.
In above-mentioned treatment CHB medicine, also have one type as ancillary drug be type of protecting the liver medicine, its clinical a large amount of uses be typically the silymarin in the seed that is present in the feverfew Herba Silybi mariani.Herba Silybi mariani is extensive use clinically, its commodity Legalon by name on market
TMGrand or the Flavobion of sharp liver
TMThis medicine effect mainly contain following some: (one) free radical resisting is active: silymarin has protective effect for the hepatic injury that is caused by carbon tetrachloride, galactosamine, alcohols and other hepatotoxin.People such as nineteen ninety Lotteron have reported in the Mouse Liver microsome; Silymarin can reduce external lipid peroxidation that is caused by the carbon tetrachloride metabolism and the peroxidation that is caused separately by reduced coenzyme, and these show that all silymarin is the chain interruption antioxidant or is free radical scavenger.(2) protection liver plasma membrane: keep flowability of cell membranes through the anti peroxidation of lipid reaction, the protection liver plasma membrane.Can also block combining of special receptor on mycotoxin phalloidine and α-amanitin etc. and the hepatocyte, suppress it, interrupt its liver sausage circulation, thereby the enhance hepatocyte film be for the resistance of multiple damage factor hepatocellular attack and transmembrane transport.(3) promote hepatocellular reparation and regeneration: silibinin can combine with ER after getting into cell; And make it to activate; Activated receptors can enhance hepatocyte nuclear RNA polymerase 1 activity, rna transcription is strengthened, promote enzyme and proteinic synthetic; And promote the synthetic of DNA indirectly, help hepatocellular reparation and regeneration.(4) antitumor action: various active oxygens can form 8-hydroxyl guanine by the oxidation guanine, cause DNA damage, and then cause tumor, and silibinin has also shown the effect of prevention and treatment tumor as an effective free radical resisting material.This medical instrument of the clinical trial certificate of three more than ten years has definite curative effect and hypotoxicity (to consult Flora, K. etc., Am.J.Gastroenterol, 1998,93,139-143; Saller R. etc., Drugs, 2001,61 (14), 2035-2063;
R. etc.; Curr.Med.Chem.2007; 14,315-338;
Z. etc.; Phytother.Res.2003; 17,524-530;
R. etc.; Bioorg.Med.Chem.2004; 12,5677-5687; Varga, Z. etc., Phytothe.Res.2001,15,608-612.Singh, R.P. etc., Curr.Cancer Drug Tar.2004,4,1-11).Flavanolignan's silibinin content is maximum in the Herba Silybi mariani, and activity is also the highest.Flavanolignan's chemical compound belongs to weedtree quality class, is one type of natural product of and a part flavone be combined into plain by a part phenylpropyl alcohol.Therefore; The flavanolignan's compounds that with the silibinin is representative has caused increasing concern, in the period of 2006-2009, prepares and a plurality of serial silibinin analog derivative reported also has obvious antioxidation activity (Yang Leixiang, Zhao Yu etc. like the inventor; " Design; synthesis andexamination of neuron protective properties of alkenylated and amidateddehydro-silybin derivatives ", Journal of Medicinal Chemistry, 2009; 52 (23), 7732-7752; Wang Feng; Zhao Yu etc.; " Preparation ofC-23esterified silybinderivatives and evaluation of their lipid peroxidation inhibitory and DNAprotective properties ", Bioorganic&Medicinal Chemistry, 2009; 17 (17), 6380-6389; Yang Leixiang, Zhao Yu etc., " Synthesis and Antioxidant PropertiesEvaluation of Novel Silybin Analogues "; Journal of Enzyme Inhibitionand Medicinal Chemistry; 2006,21 (4), 399-404; Or the like).In these articles of inventor report, A ring, B ring, E ring and 23 substituted flavanolignan compounds of a plurality of series that design and synthesize out through the inventor all demonstrate strong effect and catch the activity of DPPH free radical and ultra-oxygen anion free radical, antioxidant activity and protection PC12 cell activity.But above-mentioned research only limits to study the antioxidation and the cytoprotection of silibinin flavonolignan.
Though be the antioxidation curative effect that flavanolignan's chemical compound of representative has the above with the silibinin; Yet almost do not appear in the newspapers in its antiviral therapy aspect, and the compounds for treating DNA of flavanolignan viroid infection especially its new purposes that is used for anti-hepatitis virus aspect (comprise and suppress hepatitis B virus e antigen HBeAg or suppress the HBV dna replication dna) is effectively developed as yet.(2006) in the recent period; Xie Jun has reported that Combined application has the interferon of antiviral and immunomodulating dual function and the silibinin phosphatide complexes of hepatoprotective is treated the result of chronic viral hepatitis B; It is more obvious with the interferon effect than independent to find that drug combination reduces ALT, AST value in patient's body, explains that the silibinin phosphatide complexes can strengthen the effect of the treatment chronic viral hepatitis B of interferon.But similarly therapeutic scheme is still flavanolignans such as silibinin as adjuvant drug, and mainly is to be used to protect liver plasma membrane but not directly to implement antivirus action with it.(left side is state-run for the state-run grade in a left side; Liu Shuling, Xu Guili, world Chinese digests magazine; 2006 14 13 phases of volume; The 1241-1246 page or leaf) summarized the active progress of the external anti-HBV of medicinal plants composition over nearly 20 years, the multiple natural product of touching upon in the literary composition does not report that wherein any flavanolignan compounds has the active relative recording of anti-HBV.Especially silibinin class natural product and derivant thereof, it is domestic that almost nobody relates to, and the present invention has carried out structure of modification and the anti-HBV activity research that forefathers do not add attention to it.One of our purpose is: flavanolignan's lead compound of hope finding to reduce HBeAg or suppressing the HBV dna replication dna has the novelty medicine that can remove HBeAg or suppress HBV dna replication dna treatment CHB thereby it is developed further into.
To sum up, from flavanolignan, seek the reactive compound in anti-hepatitis virus field, also being about to flavanolignan's structure of modification, to make it have anti-DNA viroid activity be a brand-new field.From the lead compound challenge that forefathers did not attempt especially of wherein finding to suppress HBeAg or suppressing the HBV dna replication dna.In order to explore this field; We have prepared and silibinin structure a kind of new flavanolignan's derivant of difference to some extent; Also promptly introduce new benzyloxy substituent group and replace existing methoxyl group at the E ring; Formed the lignin flavanone alcohol compound (novel flavanolignan chemical compound) of new substituted structure on the E ring, in the hope of finding the activity and even the lead compound of unusual inhibition hepatitis B virus e antigen or inhibition HBV dna replication dna.Through the detailed Literature Consult of the inventor, up to the present, still do not have about formula (1) compounds for treating hepatitis B virus infection property disease that the present invention relates to and the report for preparing anti-hepatic-B virus medicine.Flavanolignan's formula (1) chemical compound is imitated inhibition by force for HBeAg and HBV DNA and is all belonged to beyond thought discovery, has originally very significantly, accomplishes the present invention in view of the above.
Summary of the invention
The purpose of this invention is to provide the flavanolignan shown in the formula (1) or its officinal salt and be used for preparing the new purposes that reduces hepatitis B virus e antigen or suppress the treatment hepatitis B medicine of HBV dna replication dna.
The name of formula (1) chemical compound is called: (±)-2-[2,3-dihydro-3-(3-benzyloxy-4-hydroxy phenyl)-2-methylol-1,4-benzodioxane-6]-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone.
Formula (1) chemical compound can be obtained by chemical synthesis process.
Another object of the present invention has provided a kind of pharmaceutical composition that is used to reduce hepatitis B virus e antigen or suppresses HBV dna replication dna, treatment hepatitis B, it is characterized by the perhaps mixture formed of its officinal salt and pharmaceutically acceptable auxiliaries of the formula as active component (1) chemical compound of treat effective dose by containing.Its pharmaceutical dosage form can be that tablet, capsule, injection, aerosol, suppository, membrane, drop pill, paster agent, subcutaneous planting bury agent, externally-applied liniment, oral liquid or ointment, can also adopt the known controlled release of modern pharmaceutical circle or slow release formulation or nanometer formulation.
Flavanolignan of the present invention chemical compound (1) is compared with the natural product silibinin; Characteristic with differentiation on many structures and the physico-chemical property comprises that speciality such as its hydrophobicity, armaticity, Gibbs free energy, hydrogen bond receptor, electrical, intermolecular Van der Waals force and 3D conformation all have obviously different with silibinin; And chemical compound (1) molecular weight ratio silibinin has increased 76 mass units.The ligand-receptor that above-mentioned characteristic has all determined the chemical compound three-dimensional conformation shown in the formula (1) to combine with the 3d space structure of HBeAg or HBV DNA combines complex form and combination all possibly produce bigger difference; Its binding site and binding pattern, its combination free energy etc. all can produce bigger change, thereby possibly suppress HBeAg or suppress aspect the HBV dna replication dna beyond thought effect arranged.
Uniqueness on flavanolignan's existing structure shown in the formula (1); The novelty that the research of antivirus action aspect is arranged again; And in anti-hepatitis B activity test, found to be expected to the activity of unusual inhibition hepatitis B virus e antigen become and to reduce HBeAg or suppress the HBV dna replication dna and the lead compound of treatment CHB.
We have tested the growth inhibited effect of this chemical compound to the HepG2.2.15 cell, have tested it simultaneously to the hepatitis B e antigen HBeAg of HepG2.2.15 emiocytosis and to HBV
The inhibition of dna replication dna is active.Result of the test is found: the synthetic lignin flavanone alcohol compound that obtains is (±)-2-[2 among the present invention; 3-dihydro-3-(3-benzyloxy-4-hydroxy phenyl)-2-methylol-1; 4-benzodioxane-6]-2; 3-dihydro-3; 5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone has the very significant strong inhibition activity of imitating to the hepatitis B e antigen HBeAg of HepG2.2.15 emiocytosis, its active positive control (also being clinical treatment hepatitis B virus patient one line medication) lamivudine and alpha-interferon of surpassing far away; In addition; The pharmacodynamics test result also shows: the flavanolignan's chemical compound shown in the formula (1) has great inhibitory action to duplicating of hepatitis B virus DNA (HBV-DNA), and it suppresses active to duplicating of hepatitis B viruses (HBV) DNA and just surpasses 50% than low dosage (20 mcg/ml) time.Belong to strong effect non-nucleoside and suppress the hepatitis B virus natural product; Explain all that below formula (1) chemical compound has the anti-HBV effect of highly significant, thereby can expect as reducing hepatitis B virus e antigen or suppressing the lead compound continuation exploitation that HBVDNA duplicated, treated hepatitis B.
Usefulness of the present invention is: prepared the substituted silibinin flavonolignan of benzyloxy replacement methoxyl group class ether on the E ring; And find that first this ether has the patent medicine potentiality that reduce hepatitis B virus e antigen, inhibition HBV dna replication dna, control hepatitis B virus aspect; The original new drug that reduces hepatitis B e antigen HBeAg or inhibition HBV dna replication dna for exploitation treatment hepatitis B virus original new drug, exploitation provides new material base, has potential huge social benefit and economic benefit.The present invention's characteristics again is: the present invention's synthetic starting material convenient sources, and synthetic convenient.Its preparation method is simple, and raw material sources conveniently are easy to get, and cost is low, pollutes for a short time, is beneficial to the large-scale production under the energy-saving and emission-reduction condition, and industrialization prospect is very clear and definite.
The specific embodiment
The inventor is through chemosynthesis; And obtain flavanolignan's class reactive compound that this secretion that can effectively suppress hepatitis B virus e antigen (HBeAg) can effectively suppress the HBV dna replication dna again through the chromatography means, derive its chemical constitution through integration analysis such as mass spectrum and NMR spectrums again.The inventor finds; Formula (1) chemical compound does not have obvious inhibitory action to the growth of HepG2.2.15 cell; And the secretion and the duplicating of HBV DNA of the hepatitis B virus e antigen (HBeAg) of HepG2.2.15 emiocytosis had significant inhibitory effect, point out this chemical compound to have drug safety, reduce hepatitis B virus e antigen and suppress HBV dna replication dna characteristics of high efficiency.Therefore, according to the inventor's research, the flavanolignan's chemical compound shown in the formula of the present invention (1) can be used to prepare the medicine of treatment hepatitis B virus infection property disease.
In order to understand essence of the present invention better; Use the preparation of formula (1) chemical compound below respectively and, its new purposes in pharmaceutical field is described to the HepG2.2.15 cell growth inhibition and to the inhibiting result of the test of the HBeAg and the HBV dna replication dna of HepG2.2.15 emiocytosis.Embodiment has provided partial synthesis, the structure of formula (1) chemical compound and has identified and activity data.Mandatory declaration, embodiments of the invention are to be used to explain the present invention rather than limitation of the present invention, and the simple modifications that essence according to the present invention is carried out the present invention all belongs to the present invention and requires the scope protected.
Embodiment 1:Formula (1) chemical compound (±)-2-[2,3-dihydro-3-(3-benzyloxy-4-hydroxy phenyl)-2-methylol-1,4-benzodioxane-6]-2,3-dihydro-3,5, the preparation of 7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone
The present invention has prepared the flavanolignan's chemical compound shown in the formula (1) with the de novo synthesis method, except that the method, can also directly carry out oxidative coupling reaction with the 4-hydroxy-3-methoxycinnamic alcohol of commercially available taxifolin and benzyloxy replacement, step production (a 1) chemical compound.Used instrument and reagent comprise: ultraviolet spectra is measured with Shimadzu UV-240 ultraviolet spectrophotometer; Proton nmr spectra
1H-NMR measures (tetramethylsilane ether TMS is interior mark) by INOVA type NMR spectrometer with superconducting magnet (VARIAN INOVA-400MHz); Electrospray Mass Spectrometry ESI-MS is measured by BrukerEsquire 3000+ mass spectrograph, and column chromatography is produced by Haiyang Chemical Plant, Qingdao with silica GF254 (10-40 order) with silica gel (100-200,200-300 and 300-400 order) and thin layer chromatography; Agents useful for same is analytical pure, and wherein the petroleum ether boiling range is 60-90 ℃; Thin layer preparative chromatography (PTLC) is with the aluminium foil silica gel plate of Merck company; Column chromatography adopts Sweden Amersham Pharmacia Biotech AB Company products with polydextran gel Sephadex LH-20; Reverse phase silica gel RP-18 adopts the Chromatorex product of Japanese Fuji Silysia Chemical company; MCI is a Mitsubishi chemical company product, and thin plate (TLC) detects the uviol lamp with 254nm and 365nm; Developer is with iodine vapor, 10% sulphuric acid-ethanol and phosphorus molybdenum acid solution.1.1 chemical compound 2 promptly 3,4,2 ', 4 ', the preparation of 6 '-five methoxy methoxy base chalcones:
8.4 the gram potassium hydroxide is dissolved in 100 milliliters of absolute methanols, adding was dissolved with 3 grams 2,4 after room temperature was reduced in stirring; 6-trimethoxy methoxyacetophenone and 2.3 grams 3,20 ml methanol solution of 4-dimethoxy methoxybenzaldehyde were in stirring at room 10 hours; The pressure reducing and steaming solvent adds 50 ml waters, ethyl acetate extraction in the residue; Merge organic facies, saturated common salt water washing, anhydrous sodium sulfate Na
2SO
4Drying is filtered, concentrate brown residue, get yellow oil 3.8 grams through column chromatography.Yield 77%; R
f(petroleum ether/ethyl acetate=3/1): 0.10; Proton nmr spectra
1H NMR (400MHz, deuterochloroform, δ in ppm) δ: 3.36 (unimodal, 6H, OCH
3), 3.47 (unimodal, 9H, OCH
3), 5.14 (unimodal, 4H, OCH
2O), 5.22 (unimodal, 2H, OCH
2O), 5.25 (unimodal, 4H, OCH
2O), 6.58 (unimodal, 2H, H-3 ', 5 '), 6.83 (bimodal, J=16.0Hz, 1H, H-α); 7.16 (bimodal, J=8.0Hz, 1H, H-5), 7.24 (double doublet, J=2.0,8.0Hz, 1H, H-6); (7.26 bimodal, J=16.0Hz, 1H, H-β), 7.45 (bimodal, J=2.0Hz, 1H, H-2); Electrospray Mass Spectrometry ESI-MS:m/z 507 [M+H]
+
1.2 chemical compound 3 promptly 3,4,2 ', 4 ', the preparation of 6 '-five methoxy methoxy basic ring oxygen chalcones:
Get the chemical compound that obtains in 1.1 steps 2 promptly 3,4,2 '; 4 ', 6 '-five methoxy methoxy base chalcones, 2.5 grams are dissolved in 60 ml methanol, add 4 milliliters of 3 milliliters of 2N sodium hydroxide and 30% hydrogen peroxide down in stirring; In stirring at room 5 hours, ultra-violet analysis, UV (MeOH) λ were done in sampling
MaxNm: become 209,282 by 209,330, after the absworption peak complete obiteration of 330nm, explain to react completely, the pressure reducing and steaming solvent adds 50 ml waters, ethyl acetate extraction (3 * 30 milliliters) in residue.Wash anhydrous sodium sulfate drying with saturated aqueous common salt (40 milliliters) after merging organic facies.Filter, concentrate to such an extent that yellow oil 2.4 restrains yield 88%.Product directly is used for next step reaction.
1.3 chemical compound 4 i.e. (±)-trans-3,5,7,3 ', 4 '-preparation of penta hydroxy group flavanonol:
Get the chemical compound that obtains in 1.2 steps 3 promptly 3,4,2 '; 4 ', 6 '-five methoxy methoxy basic ring oxygen chalcones, 2.4 grams are dissolved in the mixed solvent of 60 ml methanol and 20 milliliters of oxolanes, to wherein dripping 3 milliliters of concentrated hydrochloric acid; Stirred pressure reducing and steaming solvent, ethyl acetate extraction (3 * 30 milliliters) 15 minutes in 50-55 ℃; Merge after the organic facies water successively, saturated aqueous common salt (each 40 milliliters) washing, anhydrous sodium sulfate drying.Filter, concentrate, residue is made eluant with chloroform-methanol (10: 1), obtains light yellow solid 1 gram, yield 73% through column chromatography; R
f(chloroform: methanol=10: 1): 0.30; Proton nmr spectra
1H NMR (400MHz, deuterated acetone, δ inppm): 4.61 (double doublet, J=4.4,11.6Hz, 1H, H-3); 4.71 (bimodal, J=4.4Hz, 1H, 3-OH), 5.02 (bimodal, J=11.6Hz, 1H; H-2), 5.95 (bimodal, J=2.4Hz, H-6), 5.98 (bimodal, J=2.4Hz, 1H; H-8), 6.85-7.07 (multiplet, 3H, Ar-H), 11.71 (unimodal, 1H, 5-OH); Electrospray Mass Spectrometry ESI-MS:m/z 303 [M-H]
+
1.4 the preparation of chemical compound (1):
In the reaction bulb of drying nitrogen protection, adding the chemical compound 4 that obtains in the 0.6 gram step 1.3 is (±)-trans-3,5,7,3 '; 4 '-the pure and mild 3-benzyloxy of penta hydroxy group flavanone-4-hydroxyl cinnamyl alcohol 0.75 gram, with 40 milliliters of anhydrous propanones and 120 milliliters of dissolvings of anhydrous benzene, stirred 20 minutes in 55-60 ℃; Add 1.5 gram Disilver carbonates again, continue to stir 12 hours, filter; Mother solution concentrates, and gets 30.8 milligrams of buff powders, yield 28% through column chromatography.
Chemical compound (1) i.e. is (±)-2-[2,3-dihydro-3-(3-benzyloxy-4-hydroxy phenyl)-2-methylol-1,4-benzodioxane-6]-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone: buff powder; C
31H
26O
10R
fValue (chloroform: ethyl acetate: oxalic acid :=25: 1: 0.25): 0.15; Proton nmr spectra
1H-NMR (deuterated acetone, 400MHz) δ 3.50 (1H, multiplet, H-23a), 3.73 (1H, multiplet, H-23b); 4.16 (1H, multiplet, H-10), 4.68 (1H, bimodal, J=11.6Hz; H-3), 4.98 (1H, bimodal, J=8.0Hz, H-11), 5.11 (1H; Bimodal, J=11.6Hz, H-2), 5.19 (2H, unimodal ,-CH
2Bn), 5.98 (1H, unimodal, H-6), 6.00 (1H, unimodal, H-8), 6.93-7.51 (11H, multiplet, Ar-H), 11.71 (1H, unimodal, 5-OH); Electrospray Mass Spectrometry ESI-MS:m/z 557 [M-H]
+
Embodiment 2:Chemical compound (1) is to the inhibitory action of the hepatitis B e antigen (HBeAg) of HepG2.2.15 emiocytosis
2.1 cell culture:
In containing 10% inactivated fetal bovine serum, 100U/ ml penicillin and 100U/ milliliter streptomycin in the DMEM culture medium of 100 mcg/ml G418, are put 37 ℃, 5%CO with the HepG2.2.15 cell culture
2, cultivate in the incubator of 100% relative humidity.
Measure the inhibitory action of formula (1) chemical compound 2.2 adopt mtt assay to the growth of HepG2.2.15 cell:
The take the logarithm HepG2.2.15 cell of trophophase becomes 1 * 10 with culture medium with cell dilution
5Individual/milliliter, be inoculated in 96 porocyte culture plates, every hole 100 microlitres, at 37 ℃, 5%CO
2, cultivate the chemical compound (1) that adds after 24 hours with the culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 1000 mcg/ml; 200 mcg/ml, 40 mcg/ml and 8 mcg/ml, every hole 200 microlitres; Each concentration is established three multiple holes, places 37 ℃, 5%CO
2, cultivate in the incubator of 100% relative humidity, to cultivate after 72 hours, every hole adds MTT reagent 10 microlitres, continues to cultivate 4 hours, discards culture medium, and every hole adds the DMSO200 microlitre, with agitator vibration 20 minutes, under the 570nm wavelength, measures the OD value with ELIASA.With the culture hole that only adds culture medium is control wells.Suppression ratio (%)=(control wells OD value-experimental group OD value)/control wells OD value * 100%.The experiment triplicate.
Measure the inhibitory action of chemical compound to hepatitis B e antigen (HBeAg): the HepG2.2.15 cell of the trophophase of taking the logarithm becomes 1 * 10 with culture medium with cell dilution
5/ milliliter is inoculated in 96 porocyte culture plates, 100 milliliters in every hole, and at 37 ℃, 5%CO
2, cultivate the sample that adds after 24 hours with the culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 20 mcg/ml, 4 mcg/ml and 0.8 mcg/ml, every hole 200 microlitres, each concentration is established three multiple holes, places 37 ℃, 5%CO
2, cultivate in the incubator of 100% relative humidity, changed the culture medium that contains the same concentrations sample in per 4 days, with the culture medium equal-volume mixing that swaps out of the same concentration of same sample, as testing sample.With HBeAg concentration in the ELISA kit measurement culture medium, represent with P/N; With the positive contrast 1 of lamivudine (3-TC) (annotate: the lamivudine test concentrations is 100 mcg/ml, 20 mcg/ml and 4 mcg/ml); With the positive contrast 2 of alpha-interferon (annotate: the alpha-interferon test concentrations is 10000 units per ml, 5000 units per ml and 1000 units per ml).
2.3 experimental result:
Experimental result is as shown in table 1, and formula (1) chemical compound has the effect of significant inhibition hepatitis B e antigen (HBeAg).Its growth to the HepG2.2.15 cell does not have obvious inhibitory action; But the HBeAg of HepG2.2.15 emiocytosis was suppressed active in 84.1% the 8th day the time, and positive control 1 lamivudine when maximum concentration (100 mcg/ml), HBeAg to be suppressed activity be zero; Positive control 2 is an alpha-interferon only has 16.9% inhibition active to HBeAg the 8th day test concentrations the highest when (10000 units per ml), and chemical compound (1) suppresses activity to HBeAg and is higher than alpha-interferon 400% nearly when 20 mcg/ml.
Table 1. sample in the time of the 8th day to the excretory hepatitis B e antigen suppression ratio of HepG2.2.15
This embodiment presentation of results: the flavanolignan's chemical compound (±) shown in the formula (1)-2-[2; 3-dihydro-3-(3-benzyloxy-4-hydroxy phenyl)-2-methylol-1; 4-benzodioxane-6]-2,3-dihydro-3,5; 7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone has significant inhibitory effect to the hepatitis B e antigen (HBeAg) of HepG2.2.15 emiocytosis; It suppresses activity greater than 80% to HBeAg when 20 mcg/ml, 84.1% HBeAg activity inhibited is arranged under this concentration, and the positive control lamivudine when Cmax 100 mcg/ml, HBeAg to be suppressed activity still be zero; Another one positive control one line medication alpha-interferon only had 16.9% inhibition active to HBeAg on the 8th day when the highest test concentrations (10000 units per ml), and is lower about 4 times than chemical compound (1).Suppress hepatitis B virus secretion hepatitis B virus e antigen activity very significantly it is thus clear that this flavanolignan has, thereby can expect and develop into the medicine that reduces hepatitis B e antigen, control Type B viral hepatitis symptom.
Embodiment 3:The inhibitory action that formula (1) chemical compound duplicates the hepatitis B virus DNA (HBV DNA) of HepG2.2.15 emiocytosis
3.1 cell culture: method is with embodiment 2.
Measure the inhibitory action of the flavanolignan's chemical compound shown in the formula (1) to the growth of HepG2.2.15 cell 3.2 adopt mtt assay: method is with embodiment 2.
3.3 the inhibitory action that the flavanolignan's chemical compound shown in the mensuration formula (1) duplicates hepatitis B virus DNA (HBV DNA): the HepG2.2.15 cell of the trophophase of taking the logarithm becomes 1 * 10 with culture medium with cell dilution
5Individual/milliliter, be inoculated in 96 porocyte culture plates, every hole 100 microlitres, at 37 ℃, 5%CO
2, cultivate the flavanolignan's chemical compound that adds after 24 hours with shown in the formula (1) of culture medium dilution in the incubator of 100% relative humidity, concentration is respectively 20 mcg/ml, 4 mcg/ml and 0.8 mcg/ml; Every hole 200 microlitres; Each concentration is established three multiple holes, places 37 ℃, 5%CO
2, cultivate in the incubator of 100% relative humidity, changed the culture medium that contains the same concentrations sample in per 4 days, with the culture medium equal-volume mixing that swaps out of the same concentration of same sample, as testing sample.Measure HBV DNA concentration in the culture medium with HBV DNA quantitative PCR kit in the time of the 8th day.With the positive contrast 1 of lamivudine (3-TC) (test concentrations is 100,20 and 4 mcg/ml), with the positive contrast 2 of alpha-interferon (test concentrations is 10000,5000 and 1000 units per ml).
3.4 experimental result: experimental result is as shown in table 2, and flavanolignan's formula (1) chemical compound of de novo synthesis has the effect of the inhibition hepatitis B virus DNA replication of strong effect.
Table 2 sample in the time of the 8th day to the suppression ratio of the HBV dna replication dna of HepG2.2.15 cell
This embodiment presentation of results: the flavanolignan's chemical compound shown in the formula (1) has great inhibitory action to duplicating of hepatitis B virus DNA (HBV DNA), and it suppresses active to duplicating of HBV DNA and just surpasses 50% than low dosage (20 mcg/ml) time.And the positive control alpha-interferon has 38.2% inhibition active to HBV DNA the 8th day test concentrations the highest when (10000 units per ml); Chemical compound (1) suppresses active alpha-interferon when being higher than 10000 units per ml concentration to HBV DNA when 20 mcg/ml; Belong to strong effect non-nucleoside and suppress the hepatitis B virus natural product; Be worth further paying close attention to and further investigation, and can expect that optimized development is to suppress the medicine of HBV dna replication dna.
When above-mentioned description elaboration was of the present invention, the purpose that embodiment is provided simultaneously was to illustrate actual mechanical process of the present invention and meaning of the present invention.In the time of in getting into claim of the present invention and its equivalent scope, practical application of the present invention comprises all general variations, cooperates, or improves.
Claims (3)
1. be used for the purposes of preparation treatment hepatitis B medicine by the flavanolignan shown in the formula (1) or its officinal salt, it is characterized by:
The name of formula (1) chemical compound is called: (±)-2-[2,3-dihydro-3-(3-benzyloxy-4-hydroxy phenyl)-2-methylol-1,4-benzodioxane-6]-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone.
2. be used for the purposes that hepatitis B virus e antigen HBeAg medicine is removed in preparation by the flavanolignan shown in the formula (1) or its officinal salt, it is characterized by:
The name of formula (1) chemical compound is called: (±)-2-[2,3-dihydro-3-(3-benzyloxy-4-hydroxy phenyl)-2-methylol-1,4-benzodioxane-6]-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone.
3. be used for the purposes that preparation suppresses hepatitis B virus DNA HBV dna replication dna medicine by the flavanolignan shown in the formula (1) or its officinal salt, it is characterized by:
The name of formula (1) chemical compound is called: (±)-2-[2,3-dihydro-3-(3-benzyloxy-4-hydroxy phenyl)-2-methylol-1,4-benzodioxane-6]-2,3-dihydro-3,5,7-trihydroxy-4H-1-.alpha.-5:6-benzopyran-4-ketone.
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